Project 1: PSD95 scaffolding of vascular K+
channels in hypertension
Shaker-type, voltage-gated K+ (KV1) channels are an
important determinant of the resting membrane potential and diameter of small
cerebral arteries. During hypertension, KV1 channel-mediated dilation appears to
be blunted and is postulated to increase myogenicity in the cerebral
circulation. However, little is known about the mechanisms that regulate the
expression of KV1 channels at the plasma membrane of cerebral vascular smooth
muscle cells (cVSMCs). In this regard, we recently identified scaffolding
proteins including PSD95 (postsynaptic density 95) in rat cVSMCs that have never
been described. PSD95 is a wellcharacterized scaffolding protein in neurons with
more than 50 known binding partners that can facilitate macromolecular signaling
between ion channels and receptors. Subsequently we determined that KV1 channels
associate with the PSD95 scaffold in cVSMCs, and that PSD95 is required for the
normal expression and dilator function of KV1 channels in small cerebral
arteries. Finally, we have evidence that the β1 adrenergic receptor (β1AR) –
another known binding partner of the PSD95 scaffold – activates a KV1
channel-mediated dilator pathway. Thus, we envision that PSD95 enables the
efficient coupling of the β1AR signaling pathway to KV1 channels in cVSMCs and
we have designed experiments to characterize the impact of this novel PSD95
complex on cerebrovascular reactivity. Based on our early findings, we
hypothesize that: β1AR and the KV1 channels form a macromolecular vasodilator
complex on a PSD95 scaffold in the rat cerebral circulation. We further propose
that the down-regulation of cerebrovascular KV1 channels during hypertension
disrupts the PSD95 scaffold resulting in a synchronized loss of the β1AR-KV1
signaling pathway and a vasodilator defect. These hypotheses will be tested
using co-immunoprecipitation and confocal microscopy to discern protein
interactions in small cerebral arteries. The physiological impact of siRNA
knockdown of PSD95 or KV1 channels in cerebral arteries in vitro and in vivo
will be evaluated using patch-clamp electrophysiology, microvessel reactivity
assays, and intravital microscopy. The findings of this project will identify
for the first time a vasodilator complex in vascular smooth muscle that is
regulated by scaffolding proteins, and will set the stage for further studies to
understand how ion channels are localized with their signaling partners in
cVSMCs.

Project 2: An ultra-fast imaging
method for monitoring blood flow in the cerebral microcirculation The etiology of brain hemorrhage is
multifactorial and can be induced by extreme alterations in many
physiological parameters that influence cerebral blood flow (CBF)
and the capacity for cerebral autoregulation, such as blood gases,
arterial blood pressure, body temperature, the autonomic nervous
system, and neuronal activity. Using animal research, this
exploratory project aims at a better understanding of the
physiological factors that influence CBF and cerebral autoregulation
and the different factors leading to cerebral hemorrhage. The main
goal of this project is to better understand the mechanisms that
would favor or trigger cerebral hemorrhage. We hypothesize that
small capillaries are the most structurally vulnerable part of the
cerebral circulation and may rupture in extreme physiological
conditions leading to cerebral hemorrhage. We think that
investigating CBF at the level of single capillaries using an
ultra-fast imaging method would provide new insight into how small
microvessels react in response to different physiological stressors
such as hypercapnia, high blood pressure and excessive neuronal
excitability. Optical imaging techniques, nowadays, have become
powerful tools for investigating CBF. Our novel method consists of
imaging and quantifying cerebral microcirculation in rat olfactory
bulb capillaries at 250-2000 frames/sec. Unlike laser line-scanning
microscopy which is relatively slow to acquire full frame images,
our method will allow to perform live imaging of red blood cell
traffic at very high frame rate. We will test whether weakening the
wall of blood vessels with ultraviolet laser irradiation in addition
to inducing extreme physiological changes can deleteriously alter
the microcirculation and result in cerebral hemorrhage. This project
also aims at finding the optimal neuroprotective conditions that may
prevent the rupture of vulnerable microvessels during extreme
physiological conditions. By seeking a better understanding of the
mechanisms leading to brain hemorrhage, the results of this research
project will allow us to develop new strategies in the prevention
and treatment of cerebral hemorrhage.